Formation of acute atherosclerotic plaques reflects the stimulation invasion of vascular smooth muscle cells (VSMCs) from the tunica media into the tunica intima. Future therapeutics to correct atherogenesis will require: 1) identification of molecules which regulate the proliferative and/or migratory activities of the VSMCs; and 2) procedures to control these regulatory steps in vivo. Since a better understanding of coronary vasculogenesis in the embryo will provide the foundation for understanding these processes in adult atherogenic vessels, our proposal addresses the regulation of VSMC growth and migration in embryonic coronary arteries. Retroviral cell lineage analyses of the avian embryonic heart have shown that: a) progenitors of coronary VSMCs arise from the epicardial anlagen and enter the tubular heart with the epicardial mantle; b) coronary vessels form by vasculogenic mechanisms, not be angiogenic sprouting from the root of the aorta. We have shown that coronary morphogenesis can be altered by the constitutive expression of exogenous genes (e.g., FGF) via retroviral-mediated gene transfer into the coronary vasculature. Using recombinant rretroviruses and microsurgical procedures established in our laboratory, we propose two projects: 1. Regulation of growth factors directing coronary VSMC-proliferation in vivo. Two sets of replication-defective retroviruses have been constructed to alter signaling of FGF and TGF-beta, both of which regulate the proliferation and vascular participation of adult VSMCs. One set of retroviruses will u- regulate growth factors or their receptors, the other will over- express mutant receptors to block or down-regulate receptor signaling. We will examine if VSMC proliferation can be inhibited by disrupting positive-mitogenic signaling (FGF) or by stimulating negative-mitogenic signaling (TGF-beta). 2. Regulation of cytoskeletal elements during VSMC cell division and cell migration. Two molecules involved in these actomyosin-dependent cellular events will be targeted in coronary VSMCs. One will be H- calponin, an actin-binding protein which is up-regulated in VSMCs within developing atherosclerotic plaques. The other is myosin light chain kinase (MLCK), a regulator of smooth muscle contraction, cytokinesis, and nonmuscle myosin-II activity during cell migration. By varying MLCK expression, it should be feasible to control VSMC proliferation and migration. Experimental designs will involve: (1) retroviral infection of the VSMCs which form the coronary arteries; (2) expression and/or disruption in embyonic hearts of the H-calponin and MLCK. Disruption experiments will involve two independent techniques: a) functional inhibition of receptor signaling by over-expression of dominant-negative mutants; and b) translational suppression of protein synthesis with anti-sense MRNA. The proposed studies will identify the mitogenic regulators of coronary vasculogenesis and provide a foundation for rational therapeutics of atherogenesis in adults.